Compositions and methods for treatment of diabetic macular edema

ABSTRACT

Disclosed herein are compositions comprising one or more antibodies that specifically bind active plasma kallikrein (e.g., human plasma kallikrein) and methods of using such compositions for the treatment of retinal diseases, such as diabetic macular edema.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/669,607, filed Mar. 26, 2015, which claims the benefit under 35U.S.C. § 119(e) of U.S. provisional application Ser. No. 61/971,170,filed Mar. 27, 2014, each of which is herein incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

Retinal diseases affect the area of the retina that serves the centralvision. While many retinal diseases share common symptoms, each hasunique characteristics.

Diabetic macular edema (DME) is a swelling of the macula caused byretinal blood vessel leakage that occurs in patients with diabetes. DMEis the major cause of vision loss in people with diabetic retinopathy.People with diabetes have a 10 percent risk of developing the DME duringtheir lifetime. DME affects up to 30% of people who have had diabetesfor more than 20 years. If left untreated, DME can result in moderate tosevere vision loss.

SUMMARY OF THE INVENTION

The present disclosure is based, in part, on studies showing that animalmodels of diabetic macular edema and retinal diseases can be treatedwith DX-2944, a Fab antibody that binds to active PKal.

Some aspects of the disclosure relate to a method for treating a retinaldisease, such as diabetic macular edema (DME), age-related maculardegeneration, retinal vein occlusion, uveitis, endophthalmitis,polypoidal choroidal vasculopathy (PCV), or any other retinal diseasepresenting with macular edema, in a subject, the method comprisingadministering (e.g., via intravitreal injection, intraocular injection,or subcutaneous injection) an effective amount of a compositioncomprising an antibody that specifically binds to active plasmakallikrein (PKal) to a subject in need thereof.

In some embodiments, the antibody does not bind to human prekallikrein.In some embodiments, the antibody specifically binds to a catalyticdomain of human PKal. In some embodiments, the antibody interacts withone or more amino acid residues in the active human PKal and inhibitsits activity by at least 50%. The one or more amino acid residues may beone or more of V410, L412, T413, A414, Q415, R416, L418, C419, H434,C435, F436, D437, G438, L439, W445, Y475, K476, V477, S478, E479, G480,D483, F524, E527, K528, Y552, D554, Y555, A564, D572, A573, C574, K575,G576, S578, T596, S597, W598, G599, E600, G601, C602, A603, R604, Q607,P608, G609, V610, and Y611. In some embodiments, the antibody binds anepitope that comprises the segment of V410-C419, H434-L439, Y475-G480,F524-K528, Y552-Y555, D572-S578, T596-R604, or Q607-Y611.

In some embodiments, the antibody inhibits the activity of the activePKal by at least 80%. In some embodiments, the antibody has an apparentKi (K_(i,app)) lower than about 1 nM. In some embodiments, the antibodyhas a K_(i,app) lower than about 0.1 nM. In some embodiments, theantibody has a K_(i,app) lower than about 0.05 nM. In some embodiments,the antibody has a binding affinity (K_(D)) for the active PKal of lessthan 10⁻⁶M. In some embodiments, the antibody preferentially binds theactive PKal as relative to a mutant of the active PKal that contains oneor more mutations at positions R551, Q553, Y555, T558, and R560.

In some embodiments, the antibody comprises a heavy chain variableregion that comprises complementarity determining region 1 (HC CDR1),complementarity determining region 2 (HC CDR2), and complementaritydetermining region 3 (HC CDR3), and wherein the HC CDR3 comprises themotif X₉₉R₁₀₀X₁₀₁G₁₀₂X₁₀₃P₁₀₄R₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁, in which: X₉₉is R or Q; X₁₀₁ is T, I, R, S, or P; X₁₀₃ is V, I, or L; X₁₀₆ is R or W;X₁₀₇ is D or N; X₁₀₈ is A, S, D, E, or V; X₁₀₉ is F or L; X₁₁₀ is D, E,or N, and X₁₁₁ is I, N, M, or S (SEQ ID NO:15). In some embodiments, X₉₉is Q and X₁₀₁ is I, R, S, or P. In some embodiments, X₁₀₆ is W and X₁₁₁is N, M, or S. In some embodiments, X₁₀₁ is I, X₁₀₈ is E, and X₁₀₃ is Ior L. In some embodiments, X₁₀₁ is I and X₁₀₃ is I or L. In someembodiments, X₁₀₃ is I or L and X₁₁₀ is D, E, or N. In some embodiments,the heavy chain variable region includes H₃₁ in the HC CDR1. In someembodiments, the heavy chain variable region includes F₂₇, F₂₉, or bothin the framework region 1 (FR1).

In some embodiments, the antibody further comprises a light chainvariable region that comprises complementarity determining region 1 (LCCDR1), complementarity determining region 2 (LC CDR2), andcomplementarity determining region 3 (LC CDR3). In some embodiments, theLC CDR2 includes K₅₀, L₅₄, E₅₅, S₅₆, or a combination thereof. In someembodiments, the light chain variable region further includes G5₇ in theframework region 3 (FR3). In some embodiments, the light chain variableincludes N₄₅ or K₄₅ in the framework region 2 (FR2).

In some embodiments, the antibody binds to the same epitope as DX-2944or competes for binding to the active PKal with DX-2944. Such anantibody may comprise a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 ofDX-2930 and a light chain (LC) CDR1, LC CDR2, and LC CDR3 of DX-2930. Insome embodiments, the antibody comprises the HC variable domain ofDX-2930 (SEQ ID NO:3) and the LC variable domain of DX-2930 (SEQ IDNO:4). In one example, the antibody is DX-2944.

In any of the methods described herein, the antibody can be afull-length antibody or an antigen-binding fragment thereof. In someembodiments, the antibody is a Fab. In some embodiments, the antibody isa human antibody or a humanized antibody.

Also within the scope of the present disclosure are (i) pharmaceuticalcompositions for use in treating a retinal disease (e.g., DME, AMD, RVO,uveitis, endophthalmitis, or PCV), the compositions comprising one ormore antibodies binding to active PKal (e.g., those described herein)and a pharmaceutically acceptable carrier, and (ii) uses of suchcompositions or antibodies for manufacturing a medicament for use intreating the retinal disease.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A shows the reduction in fluorescein angiography signal in animalstreated with an intra-ocular injection of an anti-VEGF antibody (n=5,p<0.05 by t-test).

FIG. 1B shows similar reduction in signal in animals treated withDX-2944 (n=3 for the vehicle group, n=4 for the test article group,p<0.05 by t-test).

FIG. 2A shows an exemplary photograph of an eye after laser induced CNVtreatment from a brown Norway rat treated with NaC1 vehicle.

FIG. 2B shows an exemplary photograph of an eye after laser induced CNVin a brown Norway rat treated with DX-2944.

FIG. 3 shows the amino acid sequence of the heavy chain variable region(VH) and light chain variable region (VL) of a parent antibody,M0162-A04, from which DX2930 was derived, and their alignment with thecorresponding germline VH and VL genes as indicated. Variations inM0162-A04 as compared to the germline sequences are indicated(boldfaced). The sequences in FIG. 3, from top to bottom, correspond toSEQ ID NOS: 16-18 (light V gene) and SEQ ID NOS: 19-21.

FIG. 4 shows the amino acid sequence of the catalytic domain of humanplasma kallikrein (residues 391-638 of the full length human PKal) (SEQID NO:22). The boldfaced and underlined residues refer to those that areinvolved in the interaction with the Fab fragment of DX2930 asidentified by the crystal structure discussed in Example 2 below.

FIGS. 5A-5D are a series of graphs showing the apparent kI (K_(i,app))of a number of antibody mutants derived from M0162-A04 against humanPkal, including: X135-A01 and X135-A03 (FIG. 5A), M162-A04 and X133-B02(FIG. 5B), X133-D06 and X133-F10 (FIG. 5C), and X133-G05 and M199-A08(FIG. 5D).

FIG. 6 is a series of graphs showing the apparent Ki (K_(i,app)) ofmutant X115-F02 (see Table 2 below) against wild-type PKal and a numberof PKal mutants.

FIGS. 7A-7B show the amino acid sequences of a number of PKal mutants(catalytic domain), which were produced in Pichia cells. The sequences,from top to bottom, correspond to SEQ ID NOS: 23-27.

FIG. 8 shows the effect of DX-2944 compared with anti-VEGF positivecontrol on laser CNV in brown Norway rats at day 15. The observedreduction in by fluorescein angiography signal in animals treated withan intra-ocular injection of an anti-VEGF antibody was comparable toreduction in signal observed with animals treated with DX-2944 (n=7,p<0.05 by t-test).

FIG. 9 shows the effect of DX-2944 compared with anti-VEGF positivecontrol on laser CNV in brown Norway rats at day 22. The observedreduction in by fluorescein angiography signal in animals treated withan intra-ocular injection of an anti-VEGF antibody was comparable toreduction in signal observed with animals treated with DX-2944 (n=7,p<0.05 by t-test).

DETAILED DESCRIPTION OF THE INVENTION

Millions of people suffer from varying degrees of vision loss due toretinal diseases, in which the delicate layer of tissue that lines theinside back of the eye is damaged, reducing its ability to send lightsignals to the brain. Retinal diseases may be caused by various factors,including genetic and age related factors and other diseases such asdiabetes. Diabetic retinopathy is a condition that occurs in people thathave diabetes, either Type I or Type II diabetes. Diabetic retinopathyis thought to be the result of hyperglycemia-induced damage to themicrovasculature of the retina. This damage causes retinal blood vesselsto become more permeable. In some instances, the damaged blood vesselsleak fluid, proteins, and/or lipids onto the macula, which causesswelling and thickening of the macula. The swelling and thickening ofthe macula is referred to as diabetic macular edema (DME). Symptoms ofDME include blurred vision, vision distortion, and spots in the field ofvision (sometimes referred to as “floaters”).

The standard treatment for DME is laser photocoagulation. This treatmenthas undesirable side-effects including partial loss of peripheral visionand/or night vision.

The disclosure is based, in part, a study showing that antibodies thatbind to active plasma kallikrein (PKal) are therapeutically effective inan animal model of retinal diseases such as DME, AMD, RVO, uveitis,endophthalmitis, or PCV. Accordingly, in some aspects the disclosurerelates to compositions and methods for the treatment of a retinaldisease such as DME, AMD, RVO, uveitis, endophthalmitis, or PCV usingantibodies capable of binding to active PKal (e.g., active human PKal).

Antibodies Binding to Active PKal

The present disclosure provides isolated antibodies that specificallybind active PKal, e.g., the catalytic domain of the PKal. In someembodiments, the antibody described herein does not bind toprekallikrein (e.g., human prekallikrein).

Plasma kallikrein is a serine protease component of the contact system(Sainz I. M. et al., Thromb Haemost 98, 77-83, 2007). The contact systemis activated by either factor XIIa upon exposure to foreign ornegatively charged surfaces or on endothelial cell surfaces byprolylcarboxypeptidases (Sainz I. M. et al., Thromb Haemost 98, 77-83,2007). Activation of plasma kallikrein amplifies intrinsic coagulationvia its feedback activation of factor XII and enhances inflammation viathe production of the proinflammatory nonapeptide bradykinin. As theprimary kininogenase in the circulation, plasma kallikrein is largelyresponsible for the generation of bradykinin in the vasculature.

Exemplary plasma kallikrein sequences can include human, mouse, or ratplasma kallikrein amino acid sequences, a sequence that is 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, ora fragment thereof, e.g., of a sequence provided below.

An exemplary sequence of a mature human plasma kallikrein is shown below(see, e.g., Tang et al. (2005) Expression, Crystallization, andThree-dimensional Structure of the Catalytic Domain of Human PlasmaKallikrein. J of Biol Chem. 280(49): 41077-41089, which is incorporatedherein by reference). This exemplary sequence comprises one mutation(S⁴⁸⁴; in boldface) to facilitate production of a homogenous product.

(SEQ ID NO: 11) GCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTR/IVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPISLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDG KAQMQSPA

Factor XIIa activates prekallikrein by cleaving the polypeptide sequenceat a single site (between Arg371-Ile372, cleavage site marked by “/” inthe sequence above) to generate active plasma kallikrein, which thenconsists of two disulfide linked polypeptides; a heavy chain ofapproximately 52 kDa and a catalytic domain of approximately 34 kDa[Colman and Schmaier, (1997) “Contact System: A Vascular BiologyModulator With Anticoagulant, Profibrinolytic, Antiadhesive, andProinflammatory Attributes” Blood, 90, 3819-3843].

Exemplary human, mouse, and rat prekallikrein amino acid sequences(including signal peptides) are illustrated below. The sequences ofprekallikrein are the same as plasma kallikrein, except that activeplasma kallikrein (pKal) has the single polypeptide chain cleaved at asingle position (indicated by the “/”) to generate two chains. Thesequences provided below are full sequences that include signalsequences. On secretion from the expressing cell, it is expected thatthe signal sequences are removed.

Human plasma kallikrein (ACCESSION: NP_000883.2) >gi|78191798|ref|NP_000883.2| plasma kallikrein B1 precursor [Homo sapiens] (SEQ ID NO: 12)MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTR/IVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQS PAMouse plasma kallikrein (ACCESSION: NP_032481.1) >gi|6680584|ref|NP_032481.1| kallikrein B, plasma 1 [Mus musculus] (SEQ ID NO: 14)MILFNRVGYFVSLFATVSCGCMTQLYKNTFFRGGDLAAIYTPDAQYCQKMCTFHPRCLLFSFLAVTPPKETNKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQISACHRDIYKGLDMRGSNFNISKTDNIEECQKLCTNNFHCQFFTYATSAFYRPEYRKKCLLKHSASGTPTSIKSADNLVSGFSLKSCALSEIGCPMDIFQHSAFADLNVSQVITPDAFVCRTICTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIPQENAISGYSLLTCRKTRPEPCHSKIYSGVDFEGEELNVTFVQGADVCQETCTKTIRCQFFIYSLLPQDCKEEGCKCSLRLSTDGSPTRITYGMQGSSGYSLRLCKLVDSPDCTTKINAR/IVGGTNASLGEWPWQVSLQVKLVSQTHLCGGSIIGRQWVLTAAHCFDGIPYPDVWRIYGGILSLSEITKETPSSRIKELIIHQEYKVSEGNYDIALIKLQTPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKEQGETQNILQKATIPLVPNEECQKKYRDYVINKQMICAGYKEGGTDACKGDSGGPLVCKHSGRWQLVGITSWGEGCGRKDQPGVYTKVSEYMDWILEKTQSSDVRALETS SARat plasma kallikrein (ACCESSION: NP_036857.2) >gi|162138905|ref|NP_036857.2| kallikrein B, plasma 1 [Rattus norvegicus] (SEQ ID NO: 13)MILFKQVGYFVSLFATVSCGCLSQLYANTFFRGGDLAAIYTPDAQHCQKMCTFHPRCLLFSFLAVSPTKETDKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQLSACHQDIYEGLDMRGSNFNISKTDSIEECQKLCTNNIHCQFFTYATKAFHRPEYRKSCLLKRSSSGTPTSIKPVDNLVSGFSLKSCALSEIGCPMDIFQHFAFADLNVSHVVTPDAFVCRTVCTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIIQENAVSGYSLFTCRKARPEPCHFKIYSGVAFEGEELNATFVQGADACQETCTKTIRCQFFTYSLLPQDCKAEGCKCSLRLSTDGSPTRITYEAQGSSGYSLRLCKVVESSDCTTKINAR/IVGGTNSSLGEWPWQVSLQVKLVSQNHMCGGSIIGRQWILTAAHCFDGIPYPDVWRIYGGILNLSEITNKTPFSSIKELIIHQKYKMSEGSYDIALIKLQTPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKERGETQNILQKATIPLVPNEECQKKYRDYVITKQMICAGYKEGGIDACKGDSGGPLVCKHSGRWQLVGITSWGEGCARKEQPGVYTKVAEYIDWILEKIQSSKERALETS PA

The antibodies may be used in the methods described herein, e.g., in amethod of treating a retinal disease. The term “isolated antibody” usedherein refers to an antibody substantially free from naturallyassociated molecules, i.e., the naturally associated moleculesconstituting at most 20% by dry weight of a preparation containing theantibody. Purity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, and HPLC. In someexamples, the antibody disclosed herein specifically binds active PKalor an epitope therein.

An antibody that “specifically binds” (used interchangeably herein) to atarget or an epitope is a term well understood in the art, and methodsto determine such specific binding are also well known in the art. Amolecule is said to exhibit “specific binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular target antigen than it does withalternative targets. An antibody “specifically binds” to a targetantigen if it binds with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other substances. For example, anantibody that specifically (or preferentially) binds to human activePKal or an epitope therein is an antibody that binds this target antigenwith greater affinity, avidity, more readily, and/or with greaterduration than it binds to other antigens or other epitopes in the sameantigen. It is also understood by reading this definition that, forexample, an antibody that specifically binds to a first target antigenmay or may not specifically or preferentially bind to a second targetantigen. As such, “specific binding” or “preferential binding” does notnecessarily require (although it can include) exclusive binding.Generally, but not necessarily, reference to binding means preferentialbinding.

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (i.e., full-length) polyclonal or monoclonal antibodies,but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)₂,Fv), single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. An antibody includes an antibody of any class, suchas IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The antibodies described herein may also inhibit the activity of PKal.In some instances, the antibodies described herein can inhibit theactivity of PKal by at least 50%, e.g., 60%, 70%, 80%, 90%, 95%, orhigher. The inhibition constant (Ki) provides a measure of inhibitorpotency; it is the concentration of inhibitor required to reduce enzymeactivity by half and is not dependent on enzyme or substrateconcentrations. The inhibitory activity of an anti-PKal antibody can bedetermined by routine methods, such as the method described in Example 3below.

In some examples, the inhibitory activity of an anti-PKal antibody isdetermined by the apparent Ki (K_(i,app)) value. The K_(i,app) value ofan antibody obtained at different substrate concentrations by measuringthe inhibitory effect of different concentrations of the antibody on theextent of the reaction (e.g., enzyme activity); fitting the change inpseudo-first order rate constant as a function of inhibitorconcentration to the Morrison equation (Equation 1) yields an estimateof the apparent Ki value. For a competitive inhibitor, the Ki isobtained from the y-intercept extracted from a linear regressionanalysis of a plot of K_(i,app) versus substrate concentration.

$\begin{matrix}{v = {v_{o} - {v_{o}( \frac{( {K_{i,{app}} + I + E} ) - \sqrt{( {K_{i,{app}} + I + E} )^{2} - {4 \cdot I \cdot E}}}{2 \cdot E} )}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

In some examples, the anti-PKal antibodies described herein have aK_(i,app) value lower than 1 nM, e.g., 0.5 nM, 0.2 nM, 0.1 nM, 0.09 nM,0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM,or lower. The K_(i,app) value of an antibody can be estimated followingthe methods known in the art and described herein (Example 2).

The antibodies described herein can be murine, rat, human, or any otherorigin (including chimeric or humanized antibodies). In some examples,the antibody comprises a modified constant region, such as a constantregion that is immunologically inert, e.g., does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC). ADCC activity can be assessed using methodsdisclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constantregion is modified as described in Eur. J. Immunol. (1999) 29:2613-2624;PCT Application No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8.

Any of the antibodies described herein can be either monoclonal orpolyclonal. A “monoclonal antibody” refers to a homogenous antibodypopulation and a “polyclonal antibody” refers to a heterogeneousantibody population. These two terms do not limit the source of anantibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is ahumanized antibody. Humanized antibodies refer to forms of non-human(e.g. murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or antigen-binding fragments thereof that containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs “derived from” one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

In another example, the antibody described herein is a chimericantibody, which can include a heavy constant region and a light constantregion from a human antibody. Chimeric antibodies refer to antibodieshaving a variable region or part of variable region from a first speciesand a constant region from a second species. Typically, in thesechimeric antibodies, the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals (e.g., a non-human mammal such as mouse, rabbit, and rat), whilethe constant portions are homologous to the sequences in antibodiesderived from another mammal such as human. In some embodiments, aminoacid modifications can be made in the variable region and/or theconstant region.

In some embodiments, the anti-PKal antibodies described herein have asuitable binding affinity to a PKal or the catalytic domain thereof. Asused herein, “binding affinity” refers to the apparent associationconstant or KA. The KA is the reciprocal of the dissociation constant(K_(D)). The antibody described herein may have a binding affinity(K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower. Anincreased binding affinity corresponds to a decreased K_(D). Higheraffinity binding of an antibody to a first target relative to a secondtarget can be indicated by a higher K_(A) (or a smaller numerical valueK_(D)) for binding the first target than the K_(A) (or numerical valueK_(D)) for binding the second target. In such cases, the antibody hasspecificity for the first target (e.g., a protein in a firstconformation or mimic thereof) relative to the second target (e.g., thesame protein in a second conformation or mimic thereof; or a secondprotein). Differences in binding affinity (e.g., for specificity orother comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5,50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are inHBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) SurfactantP20). These techniques can be used to measure the concentration of boundbinding protein as a function of target protein concentration. Theconcentration of bound binding protein ([Bound]) is related to theconcentration of free target protein ([Free]) and the concentration ofbinding sites for the binding protein on the target where (N) is thenumber of binding sites per target molecule by the following equation:[Bound]=[N][Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

In some embodiments, the anti-PKal antibody comprises the heavy andlight CDRs or the heavy and light chain variable regions of DX-2930. Thesequences of the full length heavy chain and light chain of DX-2930 areshown below. The sequences of the heavy chain variable domain and thelight chain variable domain are also shown below. The sequences of theCDRs of DX-2930 are shown in Table 1.

DX-2930 Heavy Chain Amino Acid Sequence (451 amino acids) (SEQ ID NO: 1)EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGDX-2930 Light Chain Amino Acid Sequence (213 amino acids, 23419.08 Da)(SEQ ID NO: 2) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECDX-2930 Heavy Chain Variable Domain Amino Acid Sequence (SEQ ID NO: 3)EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSS DX-2930 Light Chain Variable Domain AminoAcid Sequence (SEQ ID NO: 4)DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFG QGTKVEIK

TABLE 1 CDRs for DX-2930. CDR Amino acid sequence Heavy chain CDR1HYIMM (SEQ ID NO: 5) Heavy chain CDR2 GIYSSGGITVYADSVKG (SEQ ID NO: 6)Heavy chain CDR3 RRIGVPRRDEFDI (SEQ ID NO: 7) Light chain CDR1RASQSISSWLA (SEQ ID NO: 8) Light chain CDR2 KASTLES (SEQ ID NO: 9)Light chain CDR3 QQYNTYWT (SEQ ID NO: 10)

In some embodiments, the anti-PKal antibody is a Fab comprising the sameCDRs or the heavy and light chain variable regions of DX-2930. Forexample, DX-2944, described in Example 1 below, is the Fab portion ofDX-2930.

DX-2930 is a fully human IgG derived from parent clone M0162-A04. Theamino acid sequences of the V_(H) and V_(L) of M0162-A04 are shown inFIG. 3. Their alignment with the corresponding germline VH gene(VH3_3-23) and VL gene (VK1_L12) is also shown in FIG. 3. Compared tothe HC CDR3 of M0162-A04, the HC CDR3 of DX-2930 includes the variationsof T101I, I103V, and A108E (see Table 3 below; the HC CDR3 of DX-2930being identical to M0199-A08). The Chothia Numbering Scheme is used inthe present disclosure. www.bioinf.org.uk/abs/.

Table 2 below provides structural information of DX-2930, its parentantibody M0162-A04, and variants thereof.

TABLE 2 Sequence Properties of DX-2930 Variants Name Properties M162-A04This is the parent antibody of DX-2930 that was discovered in theinitial phage display selection efforts (Ki, app = 2.5 nM). Thisantibody differs from DX-2930 at 3 critical amino acids in the CDR3 ofthe heavy chain and the germlined positions. M199-A08 Fab discoveredfollowing the affinity maturation of M0162-A04 using the Hv-CDR3 spikingmethod (Ki, app ~0.06 nM). This antibody shares the same amino acids inthe variable region with DX-2930 but was not germlined and does notcontain a Fc fragment. X115-F02 Fully human IgG, kappa light chain 1amino acid in the light chain was mutated to their germline sequence.The DNA sequence of X115-F02 was optimized for expression in CHO cellsExpressed transiently in 293T cells following subcloning into thepRH1-CHO vector DX-2930 Fully human IgG, kappa light chain 1 amino acidin the light chain and 2 amino acids in the heavy were mutated to theirgermline sequence. The DNA sequence of DX-2930 was optimized forexpression in CHO cells and cloned into the pEhl vector for stableexpression using the glutamate synthase system. The Fc of DX-2930 wasmodified to remove the C- terminal lysine reside, in order to obtain amore homogeneous product. DX-2944 This antibody is a Fab of DX-2930

Antibodies Targeting Specific Residues in Human Plasma Kallikrein

In some embodiments, the antibody that specifically binds to active PKalinteracts with one or more of the residues (e.g., at least 3, 5, 8, 10,15, 20, 25, 30, 35, 40, or 45) in the catalytic domain of human PKal,including V410, L412, T413, A414, Q415, R416, L418, C419, H434, C435,F436, D437, G438, L439, W445, Y475, K476, V477, S478, E479, G480, D483,F524, E527, K528, Y552, D554, Y555, A564, D572, A573, C574, K575, G576,S578, T596, S597, W598, G599, E600, G601, C602, A603, R604, Q607, P608,G609, V610, and/or Y611 (numbers based on the full length prekallikreinamino acid sequence). The positions of these residues are indicated inFIG. 4 (boldfaced and underlined). These residues are identified asinteracting with one or more residues in DX-2930 according to thecrystal structures described in Example 2 below.

Interacting means that the distance between two residues in a complexformed by two binding partners is lower than a predetermined value,e.g., <6 Å, <4 Å, or <2 Å. For example, an interacting residue in onebinding partner can have has at least 1 atom within a given threshold(e.g., <6 Å, <4 Å, or <2 Å) of at least 1 atom from a residue of theother binding partner on the complexed structure. Interacting does notrequire actual binding. Interacting residues are suggested as involvedin antibody recognition.

In some embodiments, the antibodies described herein bind human activePKal at an epitope comprising one or more of the residues listed above.An “epitope” refers to the site on a target compound that is bound by anantibody such as a Fab or full length antibody. An epitope can belinear, which is typically 6-15 aa in length. Alternatively, the epitopecan be conformational.

In some examples, the antibody that specifically binds to active PKaldescribed herein binds an epitope that comprises the following segments:V410-C419, H434-L439, Y475-G480, F524-K528, Y552-Y555, D572-S578,T596-R604, or Q607-Y611. In some examples, the antibody (e.g., anon-DX-2930 antibody) binds the same epitope as DX-2930 or competes forbinding to the active PKal as DX-2930.

In one example, the anti-PKal antibodies described herein preferentiallybind wild-type Pkal as compared to a mutant that includes mutations atone or more of R551, Q553, Y555, T558, and R560, e.g., Mutant 2described in Example 4. Such antibodies may bind wild-type PKal at amuch higher affinity as compared to the mutant (e.g., at least 2-fold,5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-foldhigher). Alternatively or in addition, the antibodies exhibit a muchhigher inhibitory activity against the wild-type pKal as relative to themutant (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold,200-fold, 500-fold, 1,000-fold higher).

In other examples, the anti-PKal antibodies described herein bindswild-type active PKal and functional variants thereof. The antibody canpreferentially bind an active PKal as relative to its binding to aninactive mutant. The antibody can preferentially bind active PKal asrelative to prekallikrein.

Anti-Plasma Kallikrein Antibodies Having Specific Motifs and/or Residues

In some embodiments, the anti-PKal antibody described herein comprises aV_(H) and a V_(L), each of which comprises three CDRs flanked byframework regions (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; see FIG. 3). The CDR3of the heavy chain can comprise the motif:X99R₁₀₀X₁₀₁G₁₀₂X₁₀₃P₁₀₄R₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁, in which X₉₉ is Ror Q, X₁₀₁ is T, I, R, S, or P, X₁₀₃ iS V, I, L, X₁₀₆ is R or W, X₁₀₇ isD or N, X₁₀₈ is A, S, D, E, or V, X₁₀₉ is F or L, X₁₁₀ is D, E, or N,and X₁₁₁ is I, N, M, or S (SEQ ID NO:15). In some examples, X₉₉ is Q andX₁₀₁ is I, R, S, or P. Alternatively or in addition, X106 is W and X₁₁₁is N, M, or S. In other examples, X₁₀₁ is I, X₁₀₈ is E, and X₁₀₃ is I orL; or X₁₀₁ is I and X₁₀₃ is I or L. In yet other examples, X₁₀₃ is I orL and X₁₁₀ is D, E, or N.

In addition, such an anti-pKal antibody can include one or more otherresidues that are identified based on the crystal structures discussedherein as being involved in interacting with the catalytic domain ofhuman PKal. These residues can be located in the V_(H) or the V_(L)chain. Examples include E1, V2, F27, T28, F29, and S30 in the FR1 of theV_(H), H31 in the HC CDR1; S31 and W32 in the LC CDR1, Y49 in the FR1 ofthe V_(L) chain, K50, T53, L54, and E55, and S56 in LC CDR2, and G57 andV58 the FR3 of the V_(L) chain. The anti-PKal antibodies as describedabove can use any germline heavy chain and light chain V genes as theframework. Heavy chain V genes include, but are not limited to, IGHV1-2,IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58,IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11,IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33,IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72,IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39,IGHV4-59, IGHV4-61, IGHV4-B, IGHV5-51, IGHV6-1, and IGHV7-4-1.

In some examples, the antibody uses a x light chain. Light chain VKgenes include, but are not limited to, V genes for IGKV1-05, IGKV1-06,IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27,IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8,IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29,IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11,IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41. In other examples,the antibody uses a X light chain, e.g., any of IGLV1-IGLV10.

The antibody also can use any germline heavy J segment (e.g., heavychain IGJH1-IGJH6) and light chain J segment (e.g., IGJK1, IGJK2, IGJK3,IGJK4, or IGJK5), which can subject to variations, such as deletions atthe C-terminus, N-terminus, or both.

Germline antibody gene/segment sequences are well known in the art. See,e.g., www.vbase2.org/vbstat.php.

In some examples, the anti-PKal antibody described herein uses VH3_3-23and/or VK1_L12 as the framework for the heavy chain and/or the lightchain. It may include substantially similar HC CDR1, HC CDR2, and/or HCCDR3, and LC CDR1, LC CDR2, and/or LC CDR3 as those in M0162-A04 (FIG.3), e.g., containing up to 5, 4, 3, 2, or 1 amino acid residuevariations as compared to the corresponding CDR region in M0162-A04.

In other examples, the anti-PKal antibody comprises a V_(H) chain thatincludes a V_(H) CDR1, V_(H) CDR2, and VH CDR3 at least 75% (e.g., 80%,85%, 90%, 95%, or 98%) identical to the corresponding V_(H) CDRs ofM0162-A04, and a V_(L) chain that includes a V_(L) CDR1, V_(L) CDR2, andV_(L) CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical tothe corresponding V_(L) CDRs of M0162-A04.

Alternatively, the anti-PKal antibody comprises a V_(H) chain at least75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V_(H) chain(mature or precursor) of M0162-A04 and/or a V_(L) chain at least 75%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V_(L) chain (matureof precursor) of M0162-A04.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In some instances, conservative mutations can be introduced into theCDRs in M0162-A04, e.g., at positions where the residues are not likelyto be involved in interacting with PKal as determined based on thecrystal structure. As used herein, a “conservative amino acidsubstitution” refers to an amino acid substitution that does not alterthe relative charge or size characteristics of the protein in which theamino acid substitution is made. Variants can be prepared according tomethods for altering polypeptide sequence known to one of ordinary skillin the art such as are found in references which compile such methods,e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York, 1989, or Current Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Use of Anti-PKal Antibodies for Treating Diabetic Macular Edema (DME)

Aspects of the disclosure relate to treatment of subject having,suspected of having, or at risk for having a retinal disease, forexample, DME, AMD, RVO, uveitis, endophthalmitis, or PCV. In someembodiments, methods for treating such subjects are provided, in which acomposition comprising an effective amount of an antibody thatspecifically binds to active PKal as described herein is administered tothe subject via a suitable route.

To practice a method disclosed herein, an effective amount of acomposition (e.g., a pharmaceutical composition) described herein can beadministered to a subject (e.g., a human) in need of the treatment via asuitable route, such as intravenous administration (e.g., as a bolus orby continuous infusion over a period of time), by intraocular injection,intravitreal injection, or subcutaneous injection. The composition maycomprise one or more antibodies binding to active human PKal.Alternatively, the composition may comprise nucleic acid(s) encoding theanti-PKal antibody, which may be in operable linkage to a suitablepromoter. Such a nucleic acid may be an expression vector.

The subject to be treated by the compositions and methods describedherein can be a mammal, more preferably a human, e.g., a human havingdiabetes. Mammals include, but are not limited to, farm animals, sportanimals, pets, primates, horses, dogs, cats, mice and rats. A humansubject who needs the treatment may be a human patient having, at riskfor, or suspected of having a retinal disease, including DME, AMD, RVO,uveitis, endophthalmitis, or PCV. Age-related macular degeneration (AMD)is a deterioration or breakdown of the eye's macula. With maculardegeneration, a subject may have symptoms such as blurriness, dark areasor distortion in the central vision, and optionally permanent loss ofthe central vision. Retinal vein occlusion (RVO) is a blockage of thesmall veins that carry blood away from the retina. It is often caused byhardening of the arteries (atherosclerosis) and the formation of a bloodclot. Diabetic macular edema (DME) is the proliferative form of diabeticretinopathy characterized by swelling of the retinal layers,neovascularization, vascular leak, and retinal thickening in diabetesmellitus due to leaking of fluid from blood vessels within the macula.Polypoidal choroidal vasculopathy (PCV) is a disease of the choroidalvasculature. It is present in both men and woman of many ethnicities,characterized by serosanguineous detachments of the pigmented epitheliumand exudative changes that can commonly lead to subretinal fibrosis.Uveitis is swelling and irritation of the uvea, the middle layer of theeye. The uvea provides most of the blood supply to the retina. It can becaused by autoimmune disorders, including rheumatoid arthritis orankylosing spondylitis. It can also be caused by infection or exposureto toxins. In many cases, the cause is unknown. Endophthalmitis is aninflammatory condition of the intraocular cavities (ie, the aqueousand/or vitreous humor) usually caused by infection.

A subject having such a retinal disease can be identified by routinemedical examination, e.g., a visual acuity test, tonometry, opticalcoherence tomography, color stereo fundus photography, a fluoresceinangiogram, or combinations thereof. A subject suspected of having theretinal disease might show one or more symptoms of the disease, e.g.,blurred vision, distorted vision, or spots in the field of vision. Asubject at risk for the retinal disease can be a subject having one ormore of the risk factors. For example, a subject at risk for DME mayhave one or more of the following risk factors: hypertension, fluidretention, hypoalbuminemia, or hyperlipidemia. Risk factors associatedwith RVO include atherosclerosis, diabetes, high blood pressure(hypertension), and other eye conditions, such as glaucoma, macularedema, or vitreous hemorrhage.

In some embodiments, a subject may be treated with an antibody asdescribed herein in combination with another treatment for DME.Non-limiting examples of treatment for DME include laserphotocoagulation, steroids, VEGF pathway targeting agents (e.g.,Lucentis® (ranibizumab) or Eylea® (aflibercept)), and/or anti-PDGFagents.

“An effective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size, gender and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used, that is, the highest safedose according to sound medical judgment. It will be understood by thoseof ordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of DME. Alternatively, sustained continuous releaseformulations of an anti-PKal may be appropriate. Various formulationsand devices for achieving sustained release are known in the art.

In one example, dosages for an anti-PKal antibody as described hereinmay be determined empirically in individuals who have been given one ormore administration(s) of the antibody. Individuals are givenincremental dosages of the antibody. To assess efficacy of the antibody,an indicator of a retinal disease be followed.

Generally, for administration of any of the antibodies described herein,an initial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate DME, or a symptom thereof. An exemplary dosing regimencomprises administering an initial dose of about 2 mg/kg, followed by aweekly maintenance dose of about 1 mg/kg of the antibody, or followed bya maintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Forexample, dosing from one-four times a week is contemplated. In someembodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg (such asabout 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the antibody used) can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other considerations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of ananti-PKal antibody will depend on the specific antibody (or compositionsthereof) employed, the type and severity of the retinal disease (e.g.,DME, AMD, RVO, uveitis, endophthalmitis, or PCV), whether the antibodyis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the antibody,and the discretion of the attending physician. Typically the clinicianwill administer an anti-PKal antibody, until a dosage is reached thatachieves the desired result. Administration of an anti-PKal antibody canbe continuous or intermittent, depending, for example, upon therecipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of an anti-PKal antibodymay be essentially continuous over a preselected period of time or maybe in a series of spaced dose, e.g., either before, during, or afterdeveloping the retinal disease.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has DME, a symptom of a retinal disease (e.g., DME, AMD,RVO, uveitis, endophthalmitis, or PCV), or a predisposition toward theretinal disease, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the retinal disease, thesymptom of the disease, or the predisposition toward the disease.

Alleviating a retinal disease such as DME, AMD, RVO, uveitis,endophthalmitis, or PCV, includes delaying the development orprogression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a retinal disease means to defer,hinder, slow, retard, stabilize, and/or postpone progression of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. A method that“delays” or alleviates the development of a disease, or delays the onsetof the disease, is a method that reduces probability of developing oneor more symptoms of the disease in a given time frame and/or reducesextent of the symptoms in a given time frame, when compared to not usingthe method. Such comparisons are typically based on clinical studies,using a number of subjects sufficient to give a statisticallysignificant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a retinal disease includes initial onsetand/or recurrence.

In some embodiments, the anti-PKal antibody described herein isadministered to a subject in need of the treatment at an amountsufficient to inhibit the activity of active PKal by at least 20% (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In otherembodiments, the antibody is administered in an amount effective inreducing the PKal level by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%,80%, 90% or greater).

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes intravitreal, subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, and intracranial injection orinfusion techniques. In addition, it can be administered to the subjectvia injectable depot routes of administration such as using 1-, 3-, or6-month depot injectable or biodegradable materials and methods. In someembodiments, the composition as described herein is administered into aneye of a patient where treatment is needed. In one example, it isadministered topically. In another example, it is injected intraocularlyor intravitreally.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In one embodiment, an anti-PKal antibody is administered viasite-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the anti-PKal antibody or local deliverycatheters, such as infusion catheters, an indwelling catheter, or aneedle catheter, synthetic grafts, adventitial wraps, shunts and stentsor other implantable devices, site specific carriers, direct injection,or direct application. See, e.g., PCT Publication No. WO 00/53211 andU.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338.

Therapeutic compositions containing a polynucleotide (e.g., thoseencoding the anti-PKal antibodies described herein) are administered ina range of about 100 ng to about 200 mg of DNA for local administrationin a gene therapy protocol. In some embodiments, concentration ranges ofabout 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg toabout 500 μg, and about 20 μg to about 100 μg of DNA or more can also beused during a gene therapy protocol.

Anti-PKal antibodies described herein can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51;Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy(1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters and/or enhancers. Expression of the codingsequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history. In some embodiments, more than oneanti-PKal antibodies, or a combination of an anti-PKal antibody andanother suitable therapeutic agent, may be administered to a subject inneed of the treatment. The antagonist can be the same type or differentfrom each other. The anti-PKal antibody can also be used in conjunctionwith other agents that serve to enhance and/or complement theeffectiveness of the agents.

Treatment efficacy for a retinal disease can be assessed by methodswell-known in the art, e.g., by fluorescein angiography.

Antibody Preparation

Antibodies capable of binding PKal as described herein can be made byany method known in the art. See, for example, Harlow and Lane, (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork.

In some embodiments, antibodies specific to a target antigen (e.g., ahuman PKal or the catalytic domain thereof) can be made by theconventional hybridoma technology. The full-length target antigen or afragment thereof, optionally coupled to a carrier protein such as KLH,can be used to immunize a host animal for generating antibodies bindingto that antigen. The route and schedule of immunization of the hostanimal are generally in keeping with established and conventionaltechniques for antibody stimulation and production, as further describedherein. General techniques for production of mouse, humanized, and humanantibodies are known in the art and are described herein. It iscontemplated that any mammalian subject including humans or antibodyproducing cells therefrom can be manipulated to serve as the basis forproduction of mammalian, including human hybridoma cell lines.Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-PKal monoclonal antibodies described herein.The hybridomas are expanded and subcloned, if desired, and supernatantsare assayed for anti-immunogen activity by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies capable of interfering with the PKal activity.Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a target antigen or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example maleimidobenzoyl sulfosuccinimide ester (conjugation throughcysteine residues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOLI, or R1N=C=NR, where R and R1are different alkyl groups, can yield a population of antibodies (e.g.,monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g.,produced by a hybridoma) may be sequenced and the polynucleotidesequence may then be cloned into a vector for expression or propagation.The sequence encoding the antibody of interest may be maintained invector in a host cell and the host cell can then be expanded and frozenfor future use. In an alternative, the polynucleotide sequence may beused for genetic manipulation to “humanize” the antibody or to improvethe affinity (affinity maturation), or other characteristics of theantibody. For example, the constant region may be engineered to moreresemble human constant regions to avoid immune response if the antibodyis used in clinical trials and treatments in humans. It may be desirableto genetically manipulate the antibody sequence to obtain greateraffinity to the target antigen and greater efficacy in inhibiting theactivity of PKal. It will be apparent to one of skill in the art thatone or more polynucleotide changes can be made to the antibody and stillmaintain its binding specificity to the target antigen.

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse® fromAmgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC MouseT™ fromMedarex, Inc. (Princeton, N.J.). In another alternative, antibodies maybe made recombinantly by phage display or yeast technology. See, forexample, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150;and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455, and.Alternatively, the phage display technology (McCafferty et al., (1990)Nature 348:552-553) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab∝)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies,chimeric antibodies, single-chain antibodies, Fabs, and bi-specificantibodies, can be produced via, e.g., conventional recombinanttechnology. In one example, DNA encoding a monoclonal antibodiesspecific to a target antigen can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoone or more expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. See, e.g., PCT Publication No. WO87/04462. The DNA can then be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison et al., (1984) Proc.Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, genetically engineeredantibodies, such as “chimeric” or “hybrid” antibodies; can be preparedthat have the binding specificity of a target antigen.

Techniques for producing Fabs are also known in the art (see, e.g., PCTPublication Nos. WO1993006217 and WO2005038031, which are incorporatedby reference herein). A variety of host-expression vector systems may beutilized to recombinantly express a Fab. Such host-expression systemsrepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express a Fab described herein.These include, but are not limited to, microorganisms such as bacteria(e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining coding sequences encoding a Fab antibody described herein;yeast (e.g., Saccharomyces pichia) transformed with recombinant yeastexpression vectors containing sequences encoding a Fab antibodydescribed herein; insect cell systems infected with recombinant virusexpression vectors (e.g., baclovirus) containing the sequences encodinga Fab antibody described herein; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus(CaMV) and tobacco mosaic virus (TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing sequencesencoding a Fab antibody described herein; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells harboringrecombinant expression constructs encoding a Fab antibody describedherein. In some embodiments, a Fab described herein is recombinantlyexpressed E. coli. Once a Fab has been recombinantly expressed, it maybe purified by any method known in the art for purification ofpolypeptides or antibodies for example, by chromatography (e.g., ionexchange, affinity, or sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of polypeptides or antibodies.

Techniques developed for the production of “chimeric antibodies” arewell known in the art. See, e.g., Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in theart. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033(1989). In one example, variable regions of VH and VL of a parentnon-human antibody are subjected to three-dimensional molecular modelinganalysis following methods known in the art. Next, framework amino acidresidues predicted to be important for the formation of the correct CDRstructures are identified using the same molecular modeling analysis. Inparallel, human VH and VL chains having amino acid sequences that arehomologous to those of the parent non-human antibody are identified fromany antibody gene database using the parent VH and VL sequences assearch queries. Human VH and VL acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replacedwith the CDR regions from the parent non-human antibody or functionalvariants thereof. When necessary, residues within the framework regionsof the parent chain that are predicted to be important in interactingwith the CDR regions (see above description) can be used to substitutefor the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions. Alternatively, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can beadapted to produce a phage or yeast scFv library and scFv clonesspecific to a PKal can be identified from the library following routineprocedures. Positive clones can be subjected to further screening toidentify those that inhibits PKal activity.

Antibodies obtained following a method known in the art and describedherein can be characterized using methods well known in the art. Forexample, one method is to identify the epitope to which the antigenbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. The epitope can be a linear epitope, i.e., containedin a single stretch of amino acids, or a conformational epitope formedby a three-dimensional interaction of amino acids that may notnecessarily be contained in a single stretch (primary structure linearsequence). Peptides of varying lengths (e.g., at least 4-6 amino acidslong) can be isolated or synthesized (e.g., recombinantly) and used forbinding assays with an antibody. In another example, the epitope towhich the antibody binds can be determined in a systematic screening byusing overlapping peptides derived from the target antigen sequence anddetermining binding by the antibody. According to the gene fragmentexpression assays, the open reading frame encoding the target antigen isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of the antigen with the antibodyto be tested is determined. The gene fragments may, for example, beproduced by PCR and then transcribed and translated into protein invitro, in the presence of radioactive amino acids. The binding of theantibody to the radioactively labeled antigen fragments is thendetermined by immunoprecipitation and gel electrophoresis. Certainepitopes can also be identified by using large libraries of randompeptide sequences displayed on the surface of phage particles (phagelibraries). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays. In an additional example, mutagenesis of an antigenbinding domain, domain swapping experiments and alanine scanningmutagenesis can be performed to identify residues required, sufficient,and/or necessary for epitope binding. For example, domain swappingexperiments can be performed using a mutant of a target antigen in whichvarious fragments of the PKal polypeptide have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein (such as another member of the neurotrophin protein family). Byassessing binding of the antibody to the mutant PKal (e.g., thosemutants described in Example 2 below), the importance of the particularantigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art.

Any of the suitable methods known in the art, e.g., the epitope mappingmethods as described herein, can be applied to determine whether theanti-PKal antibody binds one or more of the specific residues/segmentsin the PKal as described herein. Further, the interaction of theantibody with one or more of those defined residues in PKal can bedetermined by routine technology. For example, a crystal structure canbe determined following the method disclosed in Example 1 below and thedistances between the residues in PKal and one or more residues in theantibody can be determined accordingly. Based on such distance, whethera specific residue in PKal interacts with one or more residues in theantibody can be determined. Further, suitable methods, such ascompetition assays and target mutagenesis assays can be applied todetermine the preferential binding of a candidate anti-PKal antibody tothe PKal as compared to another target such as a mutant PKal.

Pharmaceutical Compositions

One or more of the above-described anti-PKal antibodies can be mixedwith a pharmaceutically acceptable carrier (excipient), includingbuffer, to form a pharmaceutical composition for use in alleviating DME.“Acceptable” means that the carrier must be compatible with the activeingredient of the composition (and preferably, capable of stabilizingthe active ingredient) and not deleterious to the subject to be treated.Pharmaceutically acceptable excipients (carriers) including buffers,which are well known in the art. See, e.g., Remington: The Science andPractice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins,Ed. K. E. Hoover. In one example, a pharmaceutical composition describedherein contains more than one anti-PKal antibodies that recognizedifferent epitopes/residues of active PKal.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the anti-PKal antibody, which can beprepared by methods known in the art, such as described in Epstein, etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556. Particularly useful liposomes can be generatedby the reverse phase evaporation method with a lipid compositioncomprising phosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The anti-PKal antibody may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are known in theart, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation. Forpreparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an anti-PKalantibody with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Kits for Use in Treating Retinal Diseases

The present disclosure also provides kits for use in treating a retinaldisease, such as DME, AMD, RVO, uveitis, endophthalmitis, or PCV. Suchkits can include one or more containers comprising an anti-PKalantibody, e.g., any of those described herein, for example, DX-2944.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of theanti-PKal antibody to treat, delay the onset, or alleviate a retinaldisease. The kit may further comprise a description of selecting anindividual suitable for treatment based on identifying whether thatindividual has or is at risk for the retinal disease. In still otherembodiments, the instructions comprise a description of administering anantibody to an individual at risk of the target disease.

The instructions relating to the use of an anti-PKal antibody generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as a syringe or an infusion device such as a minipump. Akit may have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The container may also have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). At least one active agent in the composition is an anti-PKalantibody as those described herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1: Effect of DX-2944 in a Laser Induced ChoroidalNeovascularization (Laser CNV) Disease Model

DX-2944 is a recombinant Fab version of DX-2930 that was expressed andpurified from an E. coli expression system. The Laser CNV model is anestablished rodent model of complications associated with human retinaldiseases, such as age-related macular degeneration (AMD), retinal veinocclusions, and macular edema. The experimental design used for thisstudy is outlined below.

-   Experimental Design

Day 1: Bilateral Laser treatment to produce 3 lesions per eye

Day 3: Bilateral Intravitreal injection of test agent, vehicle or,positive control (anti-VEGF Ab)

Day 22: In-vivo fluorescein angiography

The results in FIGS. 1A-1B and FIGS. 2A-2B indicate that DX-2944 reducesobserved CNV to approximately the same extent as the positive control(an anti-VEGF antibody). The fluorescein angiography mean signal for theanti-VEGF treated group was 7023 fluorescence units, which is similar tothat observed for the DX-2944 treated group at 7071 fluorescence units.

Example 2: Identification of Critical Residues in the Catalytic Domainof Human Plasma Kallikrein Based on Crystal Structures of DX-2930-PKalComplex

The catalytic domain of human plasma kallikrein, fused with a His-tag,was expressed in insect cells and purified initially by a nickelaffinity column. The His-tag was removed from the plasma kallikrein viatrypsin digestion and the free plasma kallikrein was purified by abenzamidine affinity column, followed by a SEC column. The purifiedproduct was examined on a PAGE gel. The result indicates that thecatalytic domain of human plasma kallikrein was properly expressed andpurified.

DX-2930 was prepared via routine recombinant technology and purified. Arecombinant Fab fragment of DX-2930 was produced via routine method andpurified.

The DX-2930 Fab fragment and the catalytic domain of human plasmakallikrein were mixed at various concentrations under suitableconditions allowing formation of antibody-PKal complexes. The complexesthus formed were examined using HPLC to determine the antibody-PKalratio in the complexes. Accordingly, the suitable concentrations of boththe antibody and the PKal were identified for formation of a 1:1complex.

The antibody-PKal complex was kept under various conditions allowing forcrystallization. Diffraction analysis was performed on the crystallizedcomplex. The crystal structures (2.1 Å and 2.4 Å) were determined basedon the diffraction statistics.

According to the crystal structures, residues in the catalytic domain ofhuman PKal that are involved in the interaction with DX-2930 wereidentified. These residues are indicated (boldfaced and underlined) inFIG. 4, which provides the amino acid sequence of the catalytic domainof human PKal (residues 391-638 of human PKal).

In addition, residues in DX-2930 that interact with PKal were alsoidentified based on the crystal structure, including E1, V2, F27, T28,F29, S30, H31, R100, I101, G102, V103, P104, R105, R106, D107, G107,K108, and D111 in the heavy chain variable region, and S31, W32, Y49,K50, T53, L54, E55, S56, G57, and V58 in the light chain variableregion.

These results indicate that HC CDR3 of DX-2930 is the main region thatinteracts with PKal and a couple of residues in the HC CDR1 and FR1might also contribute to the interaction with PKal. In the light chain,the LC CDR2 region was found to contribute to the interaction.

Further, the results also indicate that variations at certain positionswith the HC CDR3 region may be allowed. For example, position 103requires small hydrophobic residues such as V or I. As another example,R106 may be replaced with W, and E108 may be replaced with S or Dwithout substantially affecting the PKal binding activity. Similarly,D110 might be replaced with E.

Example 3: Affinity Maturation Results Match Structural InformationDerived from Crystal Structure

The heavy chain variable region, particularly the HC CDR3 region, ofantibody M0162-A04 was subject to affinity maturation. Various mutantshaving amino acid variations at one or more positions in the HC CDR3region were generated and their K_(i,app) values were determinedfollowing routine methods.

Briefly, PKal and a Fab at various concentrations are incubated togetherfor 1 hour at 30° C. A substrate peptide (cleavable by PKal) is thenadded to this PKal-Fab mixture. The rate of substrate peptidecleavage/proteolysis is then measured, and plotted against theconcentrations of the Fab. This plot is then fit to the Morrisonequation, which calculates the K_(i,app) value. The results thusobtained are shown in FIGS. 5A-5D and Table 3 below:

TABLE 3 Summary of Hv-CDR3 Affinity Maturation Results Initial Ki, appSEQ Name Hv CDR3 (nM) ID NO: M0162-A04 RRTGIPRRDAFDI    2.5 28 M0199-A11--R----------    2 29 M0201-F11 --S----------    3 30 M0202-A08-------W-----    2.8 31 M0201-A06 ---------V---    3.8 32 M0202-E03-----------E-    2 33 M0199-B01 ------------N    1.6 34 M0200-B01------------S    3.6 35 M0201-H06 ----V--------    0.6 36 M0202-H05----V----V---    0.26 37 M0201-H08 ----V-----L-N    0.8 38 M0200-E11----V-------N    0.4 39 M0200-H07 ----V---N---N    0.4 40 M0202-F06----V--W-----    0.33 41 M0200-A10 ----V----S---    0.25 42 M0202-G03----V----S-E-    0.4 43 M0202-A12 Q---V----S-N-    0.1 44 M0202-H03----V--W-D---    0.1 45 M0201-A07 ----V----E---    0.1 46 M0202-C02--P-V--------    0.6 47 M0202-B04 --S-V--------    0.2 48 M0202-E06--R-V----D---    0.06 49 M0202-A01 --I-V--------    0.3 50 M0202-D09--I-V----S---    0.2 51 M0200-D03 --I-V----S--M    0.1 52 M0202-C09--I-V----D---    0.06 53 M0199-A08 --I-V----E---    0.06  7 X133-B02--I----------    2.2 54 X133-D06 --I------E---    0.33 55 X135-A01----A--------  247.7 56 X133-G05 ----S-------- 1405.6 57 X133-F10----L--------   14.7 58 X135-A03 ---------E---    1.1 59

The affinity maturation results indicate that variations at certainpositions within the HC CDR3 region result in high affinity/inhibitoryanti-PKal antibodies as compared to the parent M0162-A04 clone. Theseresults match with the structural information provided in Example 2above. Note that the HC CDR3 region of clone M0199-A08 is identical tothat of DX-2930.

Example 4: Impact of Mutations in Plasma Kallikrein on AntibodyInhibitory Activity

The inhibitory activities of mutant X115-F02 against various PKalmutants were examined.

X115-F02 is an IgG that is the same as DX-2930 except that it contains aC-terminal lysine residue not present in DX-2930 and was expressed inHEK293T cells rather than CHO cells (Table 2 above). The bindingspecificity and affinity of X115-F02 is the same as DX-2930.

The wild type and four mutants of plasma kallikrein used in this study(FIGS. 7A-7B) are recombinant catalytic domains expressed and purifiedfrom Pichia pastoris. Mutant 1 contains the following mutations in theS3 subsite of the active site: S478A, N481A, S506A, Y507A) (numbersbased on the full length prekallikrein amino acid sequence). Mutant 2contains the following mutations in the S1′ subsite of the active site:R551A, Q553A, Y555A, T558A, R560A. Mutant 4 contains the followingmutations that are distal from the active site: N396A, S398A, W399A.Mutant 3 was found to be inactive and therefore was not tested in theactivity assay. Mutant 3 contains the following mutations in the S1′subsite of the active site: D572A, K575A, D577A.

The inhibitory activity of X115-F02 against the wild-type PKal and themutants were carried out using the method described in Example 3 aboveand the K_(l)a_(pp) values were determined. As shown in FIG. 6, themutations in Mutant 1 and 4 did not significantly affect the potency ofX115-F02 inhibition of plasma kallikrein. Surprisingly, the mutations inMutant 2 reduced the potency approximately 65-fold. These resultsindicate that residues R551A, Q553A, Y555A, T558A, R560A and theiradjacent residues might be important to the inhibitory activity ofX115-F02 (DX-2930).

Example 5: Effect of DX-2944 in a Laser Induced ChoroidalNeovascularization (Laser CNV) Disease Model—Study 3

DX-2944 as described herein was expressed and purified from an E. coliexpression system. The Laser CNV model used in this study is anestablished rodent model of complications associated with human retinaldiseases, such as age-related macular degeneration (AMD), retinal veinocclusions, and macular edema. The experimental design conducted issummarized below.

Experimental Design: Laser-induced Choroidal Neovascularization (CNV) inrats

Day 1: Bilateral Laser treatment to produce 3 lesions per eye

Day 3: Bilateral intravitreal injection of test agent, vehicle, andpositive control

Day 10: Bilateral intravitreal injection of test agent, vehicle, andpositive control

Day 15: In-vivo fluorescein angiography

Day 22: In-vivo fluorescein angiography

The results shown in FIG. 8 indicate that DX-2944 reduced observed CNVto approximately the same extent as the positive control (an anti-VEGFantibody) at Day 15 of the study. The fluorescein angiography meansignal for the anti-VEGF treated group was 4627 fluorescence units,which was similar to that observed for the DX-2944 treated group at 4917fluorescence units. The results shown in FIG. 9 indicate that DX-2944reduced observed CNV to approximately the same extent as the positivecontrol at Day 22 of the study. The fluorescein angiography mean signalfor the anti-VEGF treated group was 4551 fluorescence units, which wassimilar to that observed for the DX-2944 treated group at 5011fluorescence units.

These results show that DX-2944 was effective to reduce CNV in theanimal model, indicating that this antibody would be effective intreating human retinal diseases, such as age-related maculardegeneration (AMD), retinal vein occlusions, and macular edema.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of examples only and that, within the scope of the appendedclaims and equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method for treating a retinal disease in asubject, the method comprising: administering an effective amount of acomposition comprising an antibody to the subject, wherein the antibodycomprises: a heavy chain variable region comprising a complementaritydetermining region (CDR) 1 set forth as HYIMM (SEQ ID NO: 5), a CDR2 setforth as GIYSSGGITVYADSVKG (SEQ ID NO: 6), and a CDR3 set forth asRRTGVPRWDDFDI (SEQ ID NO: 45); and a light chain variable regioncomprising a CDR1 set forth as RASQSISSWLA (SEQ ID NO: 8), a CDR2 setforth as KASTLES (SEQ ID NO: 9), and a CDR3 set forth as QQYNTYWT (SEQID NO: 10); wherein the retinal disease is selected from the groupconsisting of diabetic macular edema (DME), age-related maculardegeneration (AMD), and retinal vein occlusion (RVO).
 2. The method ofclaim 1, wherein the retinal disease is diabetic macular edema.
 3. Themethod of claim 1, wherein the antibody is a full-length antibody or anantigen-binding fragment thereof.
 4. The method of claim 1, wherein theantibody is a Fab.
 5. The method of claim 1, wherein the antibody is ahuman antibody or a humanized antibody.
 6. The method of claim 1,wherein the composition is administered via intravitreal injection. 7.The method of claim 1, wherein the antibody is the only active agentadministered to the subject for treating the retinal disease.